The major fatty acids of a novel species of Thermogemmatispora sp. (strain T81) from the phylum Chloroflexi were identified as i18:0 (42.8 % of total fatty acids), i19:0 (9.7 %), and i17:0 (5.9 %). Also observed was a number of unidentified fatty acids, including a major acid (16.3 %) with ECL of 19.04 (BP1), and 18.76 (TG-WAXMS A). GCMS revealed that this compound is a saturated 20-carbon atom fatty acid. (1)H- and (13)C-NMR, with (1)H-(1)H-COSY and (1)H-(13)C-HSQC experiments suggested the structure of dimethyl octadecanoic acid with iso-branching, and an extra middle-chain methyl group. A pyrrolidide derivative demonstrated the characteristic gaps in GCMS indicating methyl branching at C12 and C17, which was eventually confirmed by a (1)H-(13)C-HSQC-TOCSY experiment. This 12,17-dimethyloctadecanoic acid has not been previously detected or described in these organisms. However, a recent description of a phylogenetically related species of Thermogemmatispora (Yabe et al., Int J Syst Evol Microbiol 61:903-910, 2010), noted an unidentified 20:0 fatty acid with matching GC behavior and GCMS data to that of strain T81. These data suggest that Thermogemmatispora share an ability to synthesize the same fatty acid. A number of other dimethyl-branched fatty acids, namely 8,14-diMe 15:0; 12,15-diMe 16:0; 10,15-diMe 16:0; 12,16-diMe 17:0; 10,16-diMe 17:0; 12,17-diMe 18:0; 12,18-diMe 19:0; 14,19-diMe 20:0, were also identified in strain T81.